This invention concerns neurotrophic compounds and agents useful in the treatment of neurological injury and disease.
Following traumatic or mechanically induced axonal degeneration in the peripheral nervous system, axonal regeneration often ensues, resulting in functional recovery. However, the rate of axonal elongation (3-4 mm/day) is slow, and sometimes does not result in recovery of full neurological function. If neurological function is restored, recovery usually occurs in weeks or months, depending upon the distance between the site of injury and the target tissue. Therapies that speed regeneration over long distances would be highly beneficial to patients and would significantly reduce health care costs.
Other neurological conditions result from dysfunction of neurons in the peripheral or central nervous systems that is caused by chronic disease or injury. Chronic disease processes can permanently and progressively damage the nervous system, and (particularly in the central nervous system) usually results in permanent loss of function. Such loss of neurological function is a major cause of physical incapacitation and death throughout the world.
The immunosuppressant drug FK506 (USAN tacrolimus; Prograf(copyright)) induces immunosuppression by binding the immunophilin FKBP-12. This binding prevents calcineurin from dephosphorylating the transcription factor NF/AT (nuclear factor of activated T-cells), which blocks translocation of calcineurin into the nucleus, and prevents a receptor-mediated increase in the synthesis and secretion of cytokines, such as interleukin-2 (IL-2), which are required for T-cell proliferation. FK506 has also been found to speed functional recovery and axonal regeneration in the rat in a dose-dependent manner following a sciatic nerve crush lesion.
U.S. Pat. No. 5,654,332 (Armistead et al.) discusses immunosuppressive FK506 analogs that bind FKBP-12, and are said to stimulate neurite outgrowth in the presence of NGF. The neurotrophic activity of these FKBP-12 binding compounds was said to be xe2x80x9cdirectly related to their affinity for FKBP-12 and their ability to inhibit FKBP-12 rotamase activityxe2x80x9d (id. at col. 7, lines 47-50). Rotamase activity measures peptidylisomerase cis-trans isomerization, and inhibition of this activity has been accepted as an indication of the immunosuppresant and neurotrophic activity of therapeutic agents. See U.S. Pat. No. 5,614,547 (Hamilton et al.).
Systemic administration of two synthetic FK506 analogs that bind FKBP-12 but that do not inhibit calcineurin activity (and which are not immunosuppressants) have been reported to increase the size of myelinated fibers (Gold et al., Exp. Neurol. 147:269-278, 1997; Steiner et al., Nature Medicine 3:1-8, 1997; Steiner et al., Proc. Natl. Acad. Sci. USA 94:2019-2024, 1997). It has also been reported that androgens and estrogens stimulate facial nerve regeneration in hamsters (e.g. Tanzer and Jones, Exp. Neurol. 146:258-264, 1997).
Many of the compounds previously shown to stimulate nerve regeneration have undesired side-effects, such as immunosuppression (FK506 and analogs that retain immunosuppressant activity) or androgenic or estrogenic stimulation. There is therefore a need to provide a class of nerve growth stimulating compounds that are well tolerated by subjects who take them.
The mechanism by which FK506 and other analogs induces nerve growth stimulation has previously been misunderstood, which has been an obstacle to the development of new drugs for this purpose.
The present invention takes advantage of the surprising discovery that nerve growth stimulation is promoted by disruption of the mature steroid receptor complex, and not by interaction with FKBP-12, as was previously thought. Disruption of the complex can include inhibition of physical assembly, promotion of disassembly, or functional interference with the steroid receptor complex, for example the mature steroid receptor complex, or a less mature form of the complex that is a predecessor to the mature complex. The participation of MAP kinase/kinase (MEK) in stimulating nerve growth, and the role heat plays in increasing neurite outgrowth when combined with nerve growth stimulating compounds, are additional parameters that can be exploited as part of this invention.
In view of the discovery of the biochemical mechanism by which neurite outgrowth is promoted, assays have been developed for selecting new compounds that may have activity in promoting nerve growth. Such assays may include determining if a test compound, other than a steroid ligand such as an androgen or an estrogen, disrupts assembly of the steroid receptor complex, and selecting a compound that disrupts assembly of the steroid receptor complex. Alternatively, the assay may include determining the ability of test compounds to stimulate MEK activity, and selecting compounds on this basis. Examples of specific classes of compounds that can be screened include geldanamycin and its structural analogs, rapamycin and its structural analogs, and FK506 and its structural analogs, radicicol and its analogs and bastadins and their analogs. Compounds selected by this assay for further investigation may be tested in additional assays to measure actual neurite outgrowth induced by the compound. In this way neurotrophic compounds have been identified by the assay for disruption of the steroid receptor complex or stimulation of MEK.
Methods have been designed for stimulating nerve cell growth in a subject by administering to the subject a compound (including a compound discovered by the assay) that disrupts assembly or function of the steroid receptor complex, for example of the mature steroid receptor complex, (for example by inhibiting association or promoting dissociation), or stimulates MEK activity, wherein the compound is other than a ligand for the steroid hormone binding portion of the steroid receptor complex (such as an androgen or an estrogen), and in some specific embodiments does not bind with high affinity to FKBP-12. A therapeutic amount of heat may be administered in combination with nerve growth stimulating compounds. In particular embodiments, the compound is administered to disrupt association of a p23 component of the steroid receptor complex with an hsp-90 component or disrupt association of FKBP-52 with hsp-90. In other embodiments, the compound is administered to competitively bind with ATP at an amino terminal ATP binding site of hsp-90, for example at a geldanamycin binding site of the steroid receptor complex. In yet other embodiments, the compound is administered to stimulate MEK activity. These methods include administration of a benzoquinone ansamycin, such as geldanamycin or a structural analog or mimetic thereof, an anti-FKBP-52 antibody, radicicol or a structural analog or mimetic thereof, or a bastadin or a structural analog or mimetic thereof. The method can also include administering a second neurotrophic factor, other than the compound that disrupts association of the steroid receptor complex.
The method is useful in the treatment of animals (including mammals such as humans) having a neurological condition associated with neuronal dysfunction caused by disease or injury to neurons in either the central or peripheral nervous systems. Compounds or compositions are administered, with or without heat, to the animal in a therapeutically effective neurotrophic amount to bind to the mature steroid receptor complex (for example at a geldanamycin binding site of hsp-90) to disrupt association of the mature steroid receptor complex or stimulate MEK activity, and promote neurite outgrowth from neurons. The method can also be used in association with procedures such as a surgical nerve graft, or other implantation of neurological tissue, to promote healing of the graft or implant, and promote incorporation of the graft or implant into adjacent tissue.
Certain pharmaceutical compounds that are not a ligand for the steroid hormone binding portion of the steroid receptor complex can disrupt assembly of a steroid receptor complex. These compounds can be geldanamycin and its structural analogs, rapamycin and its structural analogs, FK506 and its structural analogs, radicicol or a structural analog or mimetic thereof, or a bastadin or a structural analog or mimetic thereof, but more particular embodiments of the compound may have low rotamase inhibition activity, may be other than an FK506 or rapamycin analog, may not bind with high affinity to FKBP-12, or are not immunosuppressive. In particular embodiments, the compound specifically disrupts formation of the steroid receptor complex (for example the mature steroid receptor complex) either by inhibiting association or promoting dissociation of the steroid receptor complex, for example by disrupting association of a p23 component of the steroid receptor complex with an hsp-90 component, or disrupting association of FKBP-52 with hsp-90, or inhibiting interaction of p23, FKBP-52 or hsp-90 with the complex. Certain embodiments of the compound competitively bind with ATP at an amino terminal ATP binding site of hsp-90, which is also the binding site for geldanamycin binding to the steroid receptor complex. In particular embodiments the compound is radicicol or a radicicol analog that binds to a geldanamycin binding site of hsp-90. In other embodiments, the compound is an anti-FKBP-52 antibody, or another agent that specifically causes FKBP-52 to dissociate from hsp-90 of the steroid receptor complex.
The compound can be incorporated into a pharmaceutical composition, which can also include another neurotrophic factor, such as NGF, IGF-1, xcex1FGF, xcex2FGF, PDGF, BDNF, CNTF, GDNF, NT-3, NT 4/5, and mixtures thereof, or a steroid hormone that is a ligand of the steroid receptor complex (such as an estrogen, an androgen or a corticosteroid such as dexamethasone).
In a more specific aspect, the compound is a nerve growth stimulating amount of an agent that binds to a polypeptide of a steroid receptor complex other than a steroid hormone binding portion of the complex, the agent being selected from the group consisting of an FK506 analog having low binding affinity for FKBP-12 and low rotamase activity, for example a benzoquinone ansamycin and structural analogs thereof, a peptide comprising a sequence of a selected polypeptide component of the complex at a site of interaction between the selected component and another polypeptide component of the complex, an antibody, and combinations thereof, wherein the agent disrupts assembly or interferes with function of the steroid receptor complex by causing p23 or FKBP-52 dissociation from the complex, or inhibiting p23 or FKBP-52 association with the complex, or inhibiting interaction of p23, FkBP-52 or hsp-90 with the complex.
Compounds of the present invention need not have significant calcineurin inhibition or rotamase inhibition. The compounds may have an IC50 for rotamase inhibition of greater than 1 nM, for example greater than 10 nM, 25 nM, or even 50-100 nM.
Nerve cell growth can be stimulated in a subject by administering to the subject a compound that stimulates nerve cell growth, wherein the compound is one or more of radicicol or its analogs; a bastadin or its analogs; or an agent that stimulates MAP kinaseikinase activity. In particular embodiments, the compound is a radicicol analog, or a bastadin such as bastadin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, particularly bastadin 10, or an analog of a bastadin.
The method also includes administering radicicol or its analogs, or the bastadin or its analogs, in combination with a neurotrophic factor other than the compound that disrupts association of the mature steroid receptor complex or stimulates MAP kinase/kinase activity. The neurotrophic factor may be, for example, NGF, IGF-1, xcex1-FGF, xcex2-FGF, PDGF, BDNF, CNTF, GDNF, NT-3, NT 4/5, and mixtures thereof.
Another aspect is screening for agents that stimulate nerve cell growth, by detecting agents that stimulate MAP kinase/kinase activity, such as radicicol analogs, or platelet derived growth factor BB (PDGFBB) or analogs thereof.
The method also includes applying a sufficient amount of heat to an area where nerve cell growth is desired, for example along a normal anatomic pathway, or in an anatomic region, of a transected, partially transected or otherwise damaged nerve. Alternatively, the body temperature of a subject can be systemically elevated, for example by inducing a fever or placing the body in a heated environment. The invention can also include providing a template in an area where nerve growth is desired, for example a tubular member that defines an anatomical pathway along which nerve growth is desired. If desired, a therapeutically effective amount of the neurotrophic compound may be provided in association with the template to promote nerve growth. The template may be placed between opposing ends of a transected or partially transected nerve. Heat can be applied to the template in a therapeutically sufficient amount, effective to enhance nerve growth. Alternatively, the template along the desired anatomical path can be impregnated with the neurotrophic compound, or the impregnated template can be heated.
In some embodiments, the radicicol compound or its analogs are of the formula: 
where X, Y, Z, R1, R2 and R3 are as defined in Example 14, and in particular embodiments is not radicicol.
In some embodiments, the neurotrophic compound is a complete bastadin or bastadin analog, such as, 
where each R is independently selected from the group consisting of H, C1-8 alkyl, or sulfato, W is selected from the group consisting of H, OH, or C1-8 alkoxy, X, Y, and Z are independently selected from the group consisting of hydrogen, halogen, hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms that are joined by a single or a double bond. Specific complete bastadin structures (which also show the naturally occurring bastadin macrocylic ring structures) are found in Example 15.
Also included are hemibastadins and their analogs, of the formula 
where A, B, R, W, X, and Y are defined as above for the complete bastadins. Specific hemibastadin structures are found in Example 15.
Also included are hemibastadinols and their analogs of the formula 
where A, B, R, W, X, and Y are defined as above for the complete bastadins. Specific hemibastadinol structures are found in Example 15.
In other embodiments, the compound is a bastadin subunit such as 
where A, B, R, W, and X are defined as above for the complete bastadins. A specific example of a bastadin subunit is shown in Example 15.
The foregoing and various features and advantages of the invention will become more apparent from the following detailed description and accompanying drawings.